Biodetectors targeted to specific ligands

ABSTRACT

The present invention relates to biodetectors for detecting and quantifying molecules in liquid, gas, or matrices. More specifically, the present invention relates to biodetectors comprising a molecular switching mechanism to express a reporter gene upon interaction with target substances. The invention further relates to methods using such biodetectors for detecting and quantifying selected substances with high specificity and high sensitivity.

[0001] This application is a continuation-in-part of co-owned,co-pending U.S. patent application Ser. No. 08/844,336, filed Apr. 18,1997, which claims the benefit of U.S. Provisional Application No.60/015,633, filed Apr. 19, 1996.

I. FIELD OF THE INVENTION

[0002] The present invention relates to biodetectors for detecting andquantifying molecules in liquid, gas, or on solid matrices. Morespecifically, the present invention relates to biodetectors comprising amolecular switching mechanism to express a reporter gene uponinteraction with target substances. The invention further relates tomethods using such biodetectors for detecting and quantifying selectedsubstances with high specificity and sensitivity.

II. BACKGROUND OF THE INVENTION

[0003] The detection of low-levels of biological and inorganic materialsin biological samples, in the body or in the environment is frequentlydifficult. Assays for this type of detection involve multiple stepswhich can include binding of a primary antibody, several wash steps,binding of a second antibody, additional wash steps, and depending onthe detection system, additional enzymatic and washing steps. Suchassays further suffer from lack of sensitivity and are subject toinaccuracies. For instance, traditional immunoassays have false negativeresults of up to 30% when detecting infections.

[0004] Molecular probe assays, although sensitive, require highlyskilled personnel and knowledge of the nucleic acid sequence of theorganism. Both the use of nucleic acid probes and assays based on thepolymerase chain reaction (PCR) can only detect nucleic acid whichrequire complicated extraction procedures and may or may not be theprimary indicator of a disease state or contaminant. Both types of assayformats are limited in their repertoire in cases where littleinformation is available for the entity to be detected.

[0005] Current noninvasive methods to measure a patient's physicalparameters, such as CAT or MRI, are expensive and are ofteninaccessible. Thus, the monitoring of many medical problems stillrequires tests, which can be slow and expensive. The time between theactual test and the confirmation of the condition may be very important.For example, in the case of sepsis, many patients succumb beforeinfection is confirmed and the infecting organism identified, thustreatment tends to be empirical and less effective.

[0006] Another example is in screening the blood supply for pathogens.Verification of a pathogen free blood supply requires a number of laborintensive assays. In the case of HIV-1, the virus that causes AIDS, thecurrent assays screen for anti-HIV antibodies and not the virus itself.There is a window lasting up to many weeks after exposure to the virusin which antibodies are not detectable, and yet the blood contains largeamounts of infectious virus particles. Clark et al., 1994, J. Infect.Dis. 170:194-197; Piatak et al., 1993, Aids Suppl; 2:S65-71. Thus,screening of the blood supply is not only time-consuming and slow, itmay also be inaccurate.

[0007] Similarly, the ability to detect substances in the environment,such as airborne and waterborne contaminants is of great importance. Forexample, it would be desirable to monitor groundwater, to controlindustrial processes, food processing and handling in real-time using aninexpensive versatile assay. However, current methods are not suited forsuch “on-line” monitoring.

[0008] There are several reasons why current methods are limited. First,access to sufficient amounts of the material to be detected may bedifficult. For example, the detection of biological materials can bedifficult as the biological materials of interest are often sequesteredinside a body, and large quantities can be difficult to obtain for exvivo monitoring. Therefore, sensitive assays for use on small amounts ofmaterial are necessary. This indicates that a method of amplifying thesignal is required.

[0009] Amplification methods have been established for detection ofnucleic acids but this is not the case for antigen detection methods.

[0010] A second problem is that sensing may be difficult in real-timebecause the target materials may be present in such small quantitiesthat detection of their presence requires time-consuming, expensive andtechnically-involved processes. For example, in the case of bacterialinfections in the blood, sepsis, there may be only 1-2 bacteria in a1-10 ml blood sample. Current methods require that the bacteria aregrown first in order to be detected. Askin, 1995, J. Obstet. Gynecol.Neonatal. Nurs. 24:635-643. This time-lag may be detrimental as delayingtreatment or mistreating diseases may mean the difference between lifeand death.

[0011] Others have attempted to avoid these limitations by usingradioactive or fluorescent tags in combination with antibodies (Harlowet al., (1988), Antibodies. A Laboratory Manual (Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press). Antibody-based assaystypically involve binding of a primary antibody to the target molecule,followed by a series of washing steps to remove all unbound antibodies.Specific binding is typically detected using an identifier molecule,such a labeled secondary antibody directed against the primary antibody.This step is also followed by multiple wash steps. Alternatively, theprimary antibody may be directly attached to a detectable label.Suitable labels have included radioactive tracers, fluorescent tags, andchemiluminescent detection systems. Harlow, et al., 1988, Antibodies. ALaboratory Manual (Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press).

[0012] The series of steps required using such antibody-based assays togenerate a specific signal are time consuming and labor intensive.Furthermore, these type of assays are limited to the detection ofantigens fixed to some type of matrix. Examples of this type ofdetection system include Western blots, immunohistochemistry, and ELISA.The highest sensitivity is currently achieved using radioisotopic andchemiluminescent tags. However, sensitivity, i.e., specific signal overbackground, of these detection systems frequently remains a limitingfactor.

[0013] Similarly, background radiation places limits on the sensitivityof radioactive immunoassay techniques. In addition, these techniques aretime-consuming and expensive. Finally, radioactive approaches arehostile to the environment, as they present significant waste disposalproblems.

[0014] Another approach to monitoring substances involves the use oflight. Light has the advantage that it is easily measurable, noninvasiveand quantitative. Von Bally et al., (1982), Optics in BiomedicalSciences: Proceedings of the International Conference (Berlin, New York:Springer-Verlag).

[0015] Traditional spectroscopy involves shining light into substancesand calculating concentration based upon the absorbance or scattering oflight. Von Bally et al., (1982), Optics in Biomedical Sciences:Proceedings of the International Conference (Berlin, New York:Springer-Verlag). Optical techniques detect variations in theconcentration of light-absorbing or light scattering materials. VonBally et al., (1982), Optics in Biomedical Sciences: Proceedings of theInternational Conference (Berlin, New York: Springer-Verlag).Near-infrared spectroscopy has proved to be a relatively safe form ofradiation that functions well as a medical probe, since it can penetrateinto tissues. Further, it is well-tolerated in large dosages. Forexample, light is now used to calculate the concentration of oxygen inthe blood (Nellcor) or in the body (Benaron image), or even to monitorglucose in the body (Sandia). Benaron and Stevenson, 1993, Science259:1463-1466; Benaron et al., 1993, in: Medical Optical Tomography:Functional Imaging and Monitoring, G. Muller, B. Chance, R. Alfano ande. al., eds. (Bellingham, Wash. USA: SPIE Press), pp. 3-9; Benaron andStevenson, 1994, Adv. Exp. Med. Biol. 361:609-617. However, currenttechniques are limited in that many substances do not have uniquespectroscopic signals which can be optically assessed easily andquantitatively. Von Bally et al., (1982), Optics in Biomedical Sciences:Proceedings of the International Conference (Berlin, New York:Springer-Verlag). Furthermore, the detection of substances at lowconcentration is frequently hampered by high background signals,especially in biological media such as tissues. Von Bally et al.,(1982), Optics in Biomedical Sciences: Proceedings of the InternationalConference (Berlin, New York:

[0016] Springer-Verlag).

[0017] Over the past years, assays based on light emission, for examplechemiluminescence (Tatsu and Yoshikawa, 1990, Anal. Chem. 62:2103-2106),have attracted increasing attention due to the development of extremelysensitive methods for detecting and quantifying light. Hooper et al.,1994 J. Biolumin. Chemilumin. 9:113-122. One example of a biomedicalresearch product using chemiluminescence is the ECL detection system(Amersham) for immunoassays and nucleic acid detection.

[0018] The use of biological sources of light, bioluminescence, forbiological assays has paralleled development of chemiluminescentdetection, as similar devices for light detection are required. Kricka,1991, Clin. Chem. 37:1472-1481. One of the most commonly employedbiological sources of light is luciferase, a light-generating enzymesynthesized by a range of organisms, including Photinus pyralis(American firefly), Renilla reniformis (phosphorescent coral), andPhotobacterium (luminescent bacterial species). Generally, luciferase isa low molecular weight oxidoreductase, which catalyzes thedehydrogenation of luciferin in the presence of oxygen, ATP andmagnesium ions. During this process, about 96% of the energy releasedappears as visible light. For review, see, Jassim et al., 1990, J.Biolumin. Chemilumin. 5:115-122.

[0019] The sensitivity of photon detection and the ability to engineerbacteria and other cells to express bioluminescent proteins permit theuse of such cells as sensitive biosensors in environmental studies.Guzzo et al., 1992, Toxicol. Lett. 64:687-693; Heitzer et al., 1994,Appl. Environ. Microbiol. 60:1487-1494; Karube and Nakanishi, 1994,Curr. Opin. Biotechnol. 5:54-59; Phadke, 1992, Biosystems 27:203-206;Selifonova et al., 1993, Appl. Environ. Microbiol. 59:3083-3090. Forexample, Selifonova et al. describe biosensors for the detection ofpollutants in the environment. More specifically, using fusions of theHg(II) inducible Tn21 operon with the promoterless luxCDABE from Vibriofischeri, highly sensitive biosensors for the detection of Hg(II) havebeen constructed.

[0020] In addition to systems where bioluminescence is used as adetection method of a specific condition, e.g., the presence of Hg(II),supra, constitutive expression of luciferase has been employed as amarker to track viability of bacterial cells, as the luciferase assay isdependent on cell viability. For example, constitutive expression ofluciferase has recently been employed in a bacterial disease model fortesting of drugs and vaccines. Specifically, using an enhancedluciferase-expressing Mycobacterium tuberculosis strain has beenemployed to evaluate antibacterial activity in mice. Hickey et al.,1996, Antibacterial Agents and Chemotherapy 40:400-407.

[0021] However, currently-available biosensors are limited to thedetection of those molecules for which an endogenous bacterial receptorexists. In contrast, the present invention enables the generation ofbiosensors selective for any antigen or substance which can beselectively recognized by an antibody or receptor. Specifically, thepresent invention combines the selectivity of ligand-specific bindingand the versatility of the antibody repertoire with the sensitivity ofbioluminescent detection, employing entities that specifically respondwith photon emission to predetermined ligands. The approach of thepresent invention thus permits the generation of extremely sensitivebiodetectors for the development of a wide variety of assays detectingany number of commercially important molecules.

III. SUMMARY OF THE INVENTION

[0022] The present invention is directed to targeted ligand-specificbiodetectors for detecting and monitoring selected substances. Morespecifically, the biodetectors of the present invention comprise (1) asignal converting element, (2) a transducer, (3) a responsive element,and (4) a reporter gene. The signal converting element comprises anextracellular ligand-specific moiety and an intracellular signaltransforming domain. The extracellular ligand-specific moietyspecifically recognizes a selected substance. Recognition of thesubstance by the extracellular ligand-specific moiety activates theintracellular signal transforming domain. The activatedsignal-transforming domain in turn activates the transducer, which inits activated form is capable of binding to and activating theresponsive element. The responsive element, typically a promoter, isoperatively linked to the reporter gene, which encodes a polypeptidewith unique properties that are easily detected, e.g., optically. Thus,the biodetectors of the invention convert the action of binding to atarget substance, i.e., a ligand, into a detectable signal.

[0023] In a general embodiment, the signal converting element is afusion protein where the extracellular ligand-specific moiety and theintracellular signal transforming domain are heterologous to one another(i.e., are derived from different proteins). In a preferred embodiment,the extracellular ligand-specific moiety is an antibody fragment, suchas a single chain variable fragment (ScFv).

[0024] In yet another embodiment, of the invention, the intracellularsignal transforming element is derived from a membrane signal sensormolecule. The membrane signal sensor may be selected from the groupconsisting of the “sensor” of a bacterial two component regulatorysystem, a eukaryotic receptor, and a prokaryotic receptor. In a specificembodiment, the intracellular signal transforming domain comprises the3′ end of the phoQ. gene, which encodes the active or signaltransforming portion of PhoQ, the “sensor” of the PhoQ/PhoP bacterialtwo component regulatory system. In a more specific embodiment, theintracellular signal transforming domain comprises the cytoplasmic tailof PhoQ, defined as amino acids 219-487 of the PhoQ polypeptidesequence. In another specific embodiment, the intracellular signaltransforming domain comprises the cytoplasmic tail of PhoQ along withthe immediately adjacent transmembrane segment, together defined asamino acids 190-487 of the PhoQ polypeptide sequence (Miller, et al.,1989, Proc Natl Acad Sci 86:5054-5058).

[0025] In another embodiment, the signal converting element furthercomprises a membrane anchor positioned between the between theextracellular ligand-specific moiety and the intracellular signaltransforming domain. In a specific embodiment, the membrane anchor isderived from E. coli cell envelope component PAL. In yet anotherembodiment, the signal converting element further comprises anN-terminal leader sequence positioned upstream of the extracellularligand-specific moiety.

[0026] In another general embodiment, the responsive element comprises atranscription control element which is activated by the active form ofthe transducer. One embodiment of the invention thus includes promotersand/or transcription activators which control transcription intwo-component systems. In a particular embodiment, the responsiveelement comprises the phoN promoter.

[0027] In still another general embodiment, the biodetector comprises anintact bacterial ell transfected as detailed herein. In one embodiment,the biodetector is a Gram-positive bacterial cell. Such a biodetectormay be selected from the group consisting of Streptococcus,Staphylococcus, Listeria, Clostridium, Bacillus, Tuberculosis, andCorynebacteria. In another embodiment, the biodetector is aGram-negative bacterial cell. Such a biodetector may be selected fromthe group consisting of Escherichia, Salmonella, Pseudomonas,Helicobacter, Shigella, Proteus, Bordetella, Neisseria, Haemophilus,Bacteriodes, Vibrio, Brucella, Campylobacter, Rickettsia, Enterococci,Klebsiella, Spirochetes, and Yersinia. Preferred embodiments of Gramnegative biodetectors comprise Escherichia and Salmonella.

[0028] In still another embodiment of the invention, the substance thatthe biodetector is designed to detect is selected from the groupconsisting of bacteria, bacterial products, viruses, protein, sugar,lipid, liposaccharide and polysaccharide.

[0029] In another aspect, the invention includes a biodetector for thedetection of a selected substance. The biodetector comprises (a) asignal converting element, comprising an extracellular ligand-specificmoiety and an intracellular signal transforming domain, wherein theextracellular ligand-specific moiety specifically recognizes theselected substance, which recognition activates the intracellular signaltransforming domain; (b) a transducer, wherein the transducer has aninactive and an active form which are distinct from each other, andwherein the activated intracellular signal transforming domain convertsthe inactive form of the transducer into the active form of thetransducer; and (c) a responsive element, wherein the responsive elementis bound and activated by the active form of the transducer, resultingin a detectable signal.

[0030] In one embodiment, the responsive element further comprises anucleic acid encoding one or a plurality of gene product(s), which geneproduct or gene products produce the detectable signal, and wherein thenucleic acid is operatively linked to the transcription control element.In a more specific embodiment, the detectable signal is visible light.In another specific embodiment, the gene product is detectable by meansselected from the group consisting of bioluminescence, colorimetricreactions and fluorescence. In a more specific embodiment, the geneproduct is detectable by means of bioluminescence. In yet a morespecific embodiment, the nucleic acid comprises a luciferase operon.

[0031] Other specific embodiments of this aspect of the inventioninclude those summarized above for other biodetectors made in accordancewith the invention.

[0032] Also included in the invention is a library of biodetectors,where the biodetectors have characteristics, in different specificembodiments of such a library, as described above. In a generalembodiment, the biodetectors in such a library comprise a plurality ofbacterial cells transfected with a mixture of cDNA molecules encodingantibody variable region genes, Fab fragments, F(ab)₂ fragments, orsingle chain variable fragments (ScFvs). The library preferably includesat least about 1000 different biodetectors, more preferably at leastabout 10,000 different biodetectors, and even more preferably at leastabout 100,000 different biodetectors.

[0033] The present invention is further directed to methods of usingsuch biodetectors for detecting and monitoring selected substances witha high sensitivity and specificity (selectivity). The methods using thebiodetectors of the invention include the detection of contaminants inthe food and agriculture industries, diagnosis and monitoring inmedicine and research, detection of poisons or pathogenic contaminantsin the environmental or defense setting, and drug testing. Accordingly,the invention includes a method for detection of a selected substance.The method comprises the steps of (a) generating a biodetector; (b)adding the biodetector to a sample; (c) measuring and quantifying thedetectable signal; and correlating the levels of the detectable signalwith the presence and quantity of the substance. The biodetectorgenerated in step (a) comprises (i) a signal converting element,comprising an extracellular ligand-specific moiety and an intracellularsignal transforming domain, wherein the extracellular ligand-specificmoiety specifically recognizes the selected substance, which recognitionactivates the intracellular signal transforming domain; (ii) atransducer, wherein the transducer has an inactive and an active formwhich are distinct, and wherein the inactive form is converted into theactive form by the activated intracellular signal transforming domain;and (iii) a responsive element, wherein the responsive element is boundand activated by the active form of the transducer, resulting in adetectable signal.

[0034] In one embodiment, the responsive element of the biodetector inthe method comprises a transcription control element which is activatedby the active form of the transducer. In another embodiment, theresponsive element further comprises a nucleic acid encoding one or aplurality of gene products which gene product or gene products producethe detectable signal, and wherein the nucleic acid is operativelylinked to the transcription control element. In a preferred embodiment,the detectable signal is light. The gene product in such an embodimentmay be detected by a means selected from the group consisting ofbioluminescence, colorimetric reactions and fluorescence. In a specificembodiment, the nucleic acid comprises a luciferase operon. In a morespecific embodiment, the light detection system is selected from thegroup consisting of luminometer, spectrophotometer, fluorimeter, and aCCD detector.

[0035] In another embodiment, the biodetector or the sample in themethod is fixed on a solid support. In yet another embodiment, themethod further includes fixing a series of biodetectors in an orderedarray on a solid support such that a variety of substances comprised ina sample can be detected.

[0036] In another aspect, the invention includes an expression vectoruseful for making a biodetector. The vector comprises (i) a cloning sitefor insertion of a DNA fragment encoding an extracellularligand-specific moiety, and (ii) a first DNA fragment encoding anintracellular signal transforming domain. Preferably, the vector iscapable of expressing a fusion protein comprising (a) a polypeptideencoded by a DNA sequence inserted at said cloning site, and (b) theintracellular signal transforming domain. In one embodiment, the vectorfurther comprises, between the cloning site and the first DNA fragment,a second DNA fragment encoding a membrane anchor. In another embodiment,the vector further comprises, upstream of the cloning site, a third DNAfragment encoding an N-terminal leader sequence. In another embodiment,the vector further comprises, inserted at the cloning site, a fourth DNAfragment encoding an extracellular ligand-specific moiety. In anotherembodiment, the extracellular ligand-specific moiety comprises anantibody fragment. In another embodiment, the first DNA fragment encodesa polypeptide comprising the cytoplasmic tail of PhoQ.

IV. BRIEF DESCRIPTION OF THE FIGURES

[0037]FIG. 1 depicts a generic model demonstrating the main componentsof a biodetector. A biodetector consisting of an entity possessingsensing (“Y-shaped” structure on surface), transducing components (partof “Y-shaped” structure inside of biodetector) and light-emittingcomponents (small circles).

[0038]FIG. 2 depicts a more specific scheme of a biodetector on themolecular level.

[0039]FIG. 3 depicts an ordered array of biodetectors on a solid supportsuch that a variety of substances in a single sample can be detectedsimultaneously.

[0040]FIG. 4 depicts a biodetector generated by the integration of atransposon in the bacterial genome as specified in EXAMPLE 1. Thebacterial luciferase operon encodes five proteins (from genes A, B, C, Dand E) that together can produce bioluminescence. Chl, chloramphenicolresistance gene; Kan, kanamycin resistance gene; Amp, Ampicillinresistance gene; PO₄, phosphate group (as activator of the transducer).

[0041]FIG. 5 depicts the effect of human blood on the light emissionfrom bioluminescent Salmonella, demonstrating near single celldetection.

[0042]FIG. 6 depicts an expression vector according to the invention.The vector contains an antibiotic (Ampicillin) resistance gene(Amp^(r)), a promoter, a DNA sequence encoding an N-terminal leadersequence, a multiple cloning site for insertion of a DNA encoding anantibody fragment, such as a fragment comprising a variable heavy chain(V_(H)) and a variable light chain (V_(L)) region, and a DNA fragmentencoding an intracellular signal transforming domain (ISTD).

V. DEFINITIONS

[0043] Unless otherwise indicated, all terms used herein have the samemeaning as they would be understood by one skilled in the art.

[0044] The term “target molecule” as used herein describes a substancethat is to be detected and/or quantified.

[0045] The term “luciferases” as used herein, unless otherwise stated,includes prokaryotic and eukaryotic luciferases as well as variants withvaried or altered physical and/or emission properties.

[0046] The term “biodetector” as used herein refers to an entity thatresponds with an optical signal to the binding or otherwise interactingwith the target molecule.

[0047] The term “optical signal” as used herein refers to anybiochemical reaction or substance that can be distinguished using lightmonitoring techniques. This includes photon emission, fluorescence, andabsorbance.

[0048] The term “light” as used herein, unless otherwise stated, refersto electromagnetic radiation having a wavelength between about 220 nmand about 1100 nm.

[0049] The term “promoter induction” as used herein refers to an eventthat results in direct or indirect activation of a selected induciblegenetic element.

VI. DETAILED DESCRIPTION OF THE INVENTION

[0050] A. General Overview Of The Invention

[0051] The present invention is directed to targeted ligand-specificbiodetectors for detecting and monitoring selected substances, includingmicroorganisms and their products/by-products, chemical compounds,molecules, arid ions, for a wide range of applications. In a preferredembodiment, the biodetectors of the present invention combine thespecificity and selectivity of ligand-specific binding with thesensitivity of bioluminescent detection by employing entities thatspecifically respond to the binding of a predetermined ligand withphoton emission. Thus, the approach of the present invention permits thegeneration of sensitive biodetectors for the development of a widevariety of assays detecting and monitoring any selected substance.

[0052] More specifically, the biodetectors of the present inventionprovide for the coupling of ligand-specific binding, via a “molecularswitch,” i.e., a signal transduction, with the activation of adetectable reporter molecule in response to ligand binding. Thebiodetectors of the present invention may consist of viable biologicalentities, such as bacteria, or abiotic entities, such as liposomes. As ageneral scheme, the biodetectors are characterized by their ability tospecifically recognize a ligand and convert binding to the ligand to ameasurable signal, such as light emission. For example, bacteria may beemployed as ligand-specific biodetectors, which specifically respondwith photon emission to predetermined ligands.

[0053] The biodetectors of the present invention permit highly sensitivedetection of a wide variety of substances, for example microbes in humanblood (e.g., viruses and bacteria), toxic molecules, ions, cancer cells,antigens, small molecules (e.g., glucose), oxygen, and metals. Further,the present invention provides for the use of such biodetectors in awide variety of assays to detect any selected substance.

[0054] Generally, the biodetectors of the present invention comprise asignal converting element, comprising an extracellular ligand-specificbinding moiety, which is coupled to an intracellular signal transformingdomain which is capable of activating a transducer component. Thetransducer component in its active form is capable of activating aresponsive element, such as a promoter which is operatively linked to areporter gene (e.g., a luciferase gene), encoding for a diagnosticpolypeptide with unique properties that are readily detectable. Thus,the biodetectors of the invention convert the binding of a targetsubstance, i.e., a ligand, into a detectable signal. In preferredembodiments of the invention, the signal generated by the biodetector islight and is detected by a light-detecting device. Accordingly, basedupon this interaction, the targeted ligand(s) may be quantified andidentified.

[0055] B. Biodetectors

[0056] The biodetectors of the invention are characterized in that theygenerate a detectable signal in response to either the presence of atargeted substance in vivo or in vitro.

[0057] In one specific embodiment, light is the detectable signalgenerated by the biodetector in response to the presence of the targetedsubstance. As there is virtually no background light coming from normaltissues and other organic or inorganic materials, the sensitivity of thesystem is limited only by the background noise of the biodetector. Morespecifically, the ligand-specific biodetectors of the present inventioninclude a ligand-specific domain, which, via a “molecular switch,” islinked to a reporter gene encoding a detectable protein. The reportergene is thus activated in response to binding of the ligand to theligand-specific domain. The ligand-specific binding moiety may be anyantibody which selectively binds to the substance of interest. The“molecular switch” is a signal transducing component which couplesligand binding to the activation of a responsive element. Thetransducing molecule can be derived from any two component regulatorysystem of bacteria, including the phosphate regulon, or any eukaryotictransducer. The responsive element may be an inducible promoter,operatively linked to a reporter gene. Transcription and translation ofthis reporter gene will result in a gene product, e.g., luciferase,which produces a detectable signal, e.g., light. The signal is detectedby suitable means; in the case the signal is light, this means will be aphotodetection device.

[0058] For example, imaging of the light-emitting biodetector entitiesmay involve the use of a photodetector capable of detecting extremelylow levels of light—typically single photon events. If necessary,localization of signal could be determined by integrating photonemission until an image can be constructed. Examples of such sensitivephotodetectors include devices (such as microchannel plate intensifiersand photomultiplier tubes) that intensify the single photon events.Intensifiers may be placed before a camera. In addition, sensitivecameras (cooled, e.g., with liquid nitrogen) that are capable ofdetecting single photons over the background noise inherent) in adetection system may also be used.

[0059] Once a photon emission image is generated, it is typicallysuperimposed on a “normal” reflected light image of the subject toprovide a frame of reference for the source of the emitted photons. Sucha “composite” image is then analyzed to determine the location and/oramount of a target in the subject. In most circumstances images of thelight source are not required. Simple quantitation of the numbers ofphotons emitted from a sample (as detected for example by a luminometer)indicate the concentration of the light emitting reporter. The number ofphotons would therefore be proportional to the amount of targeted-ligandthat a specific detector is sensing. Without the constraints imposed bythe need for an image, detectors can be placed in very close proximityto the light-emitting biodetector thus optimizing the optical detectionand sensitivity of the assay. Microchannel plate intensifiers can beused in such a configuration resulting in single photon detection. Sucha device is currently manufactured by Hamamatsu Corporation. In theHamamatsu system ATP concentrations from single cells can be assayed byspraying lysis buffer, luciferase and the substrate, luciferin, onimmobilized cells.

[0060] The generic mechanism of a ligand-specific biodetector is shownin FIG. 1. The biodetector is combined with a gas, solution or supportmatrix that is to be tested for the presence of a selected substance orligand. If such gas, solution or support matrix contains the selectedantigen or ligand, the biodetector binds to it and generates adetectable signal, shown in FIG. 1 as light represented by outwardarrows. FIG. 2 shows the molecular mechanism of a preferred biodetectormore specifically. In the depicted example, the biodetector is abacterial cell expressing a transmembrane target specific signalconverting element, comprising an extracellular ligand-specific bindingmoiety, e.g., an antibody, which is coupled to an intracellular signaltransforming domain. The target specific signal converting element isintegrated in a membrane, e.g., a bacterial membrane, which separates an“extracellular” compartment from an “intracellular” compartment. Theligand-specific moiety (e.g., an ScFv) is capable of binding to aselected substance, which triggers the activation of the intracellularsignal transforming domain (e.g., a signaling portion of PhoQ). Theactivated intracellular signal transforming domain in turn converts aninactive transducer into an active transducer. The transducer ischaracterized by its capability to bind, when converted to its activeform, to a promoter element, which is operatively linked to a reportergene. Transcription and translation of the reporter gene or operonresults in a gene product which produces a detectable signal, such aslight. In preferred embodiments of the invention, the reporter is aluciferase gene or operon, which produces visible light and can easilybe monitored, measured and quantified with high sensitivity.Alternatively, the signal transforming domain could act directly on amodified reporter molecule. The reporter molecule would be modified tobe expressed in an inactive state which can then be activated throughits interaction with the signal transforming domain directly.

[0061] The biodetectors, providing a “light switch” that responds to apredetermined selected substance presents a number of advantages overcurrent methodologies. First, the switch allows for detection ofantigens, present in complex mixtures and eliminates the need to washoff unbound antibodies, thus simplifying the detection. Since ligandbound to antibody turns on light and since there is no background lightin the sample, no washing is necessary to reduce signal to noise ratio,reduced noise increases sensitivity, and only specific interaction turnson the light. Once bound to a ligand, an enzymatic cascade is activatedthat serves to transmit the signal.

[0062] Moreover, if the targeted ligand is abundantly expressed on thesurface of, for example, pathogenic microbes, many biodetecting bacteriawill bind to a single target, thus serving to amplify the signal andresult in extremely sensitive detection systems.

[0063] Furthermore, as the ligand-specific domain of the signalconverting element of the biodetector system may be exchanged like acassette, an unlimited number of biodetectors can be generated torecognize any desired or selected substance. Thus, the biodetectors ofthe present invention provide a flexible, generic system that can beadapted to recognize any selected substance, out of a wide variety ofchoices. Biodetectors targeting a substance of interest can rapidly bedeveloped.

[0064] The biodetectors of the invention are versatile as they areeffective in vivo, in solution, or on fixed sensor plates. Furthermore,arrays of these biodetectors may be constructed, operating at differentwavelengths or on different positions of a “biosensor chip,” allowingfor simultaneous monitoring and screening of multiple agents, genes,gene products, or other targets. See, FIG. 3. For example, thebiodetectors may be assembled in a unique multi-detector arrayconfiguration for the purpose of constructing a system capable of aplurality of parallel analyses in a single step. For example, thebiodetector may be placed on a gel that lies on top of a normal signaldetecting instrument, which, in the case the generated signal is light,may be, e.g., a charge coupled device (CCD) chip. Due to the spatialrecognition of signals by the CCD array, the biodetector array mayprovide for a light-based analysis using multiple different sensorsplaced in an array on one sensor chip. Thus, an analysis may besimultaneously performed for, e.g., blood type, HIV exposure, Hepatitisstatus, Lymphokine profiles and CMV positivity. Multiple types ofinfection can be rapidly and simultaneously screened.

[0065] If light is the signal produced by the reporter, the signal maybe detected non-invasively, as light can be detected through, forexample, tissue. See, U.S. Pat. No. 5,650,135, hereby incorporated byreference in its entirety.

[0066] Furthermore, as the biodetectors of the invention arebiocompatible, and as such environmentally friendly, they havecomparatively low developmental costs and a lower burden to the user,especially when compared with methods that may involve toxic waste, suchas radioactivity-based assays.

[0067] A further significant advantage of the biodetectors of theinvention is the reduction in time and labor needed to perform manydiagnostics tests. A common, rate-limiting step in many testing, anddiagnostic fields is the need for an accurate sensing and detectionsystem suitable for providing immediate information. Examples includescreening of the blood supply for the AIDS virus and other blood bornepathogens, the study and evaluation of novel drugs in tissue culture oranimal models, and the monitoring of therapeutic protein output aftergenetic therapy. For example, the mandatory screening of the bloodsupply for HIV and other agents currently requires numerous tests. Aninexpensive, rapid, and specific sensor detecting numerous blood bornepathogens with built-in confirmatory tests could significantlystreamline the process, thus reducing net cost to the user. Similarly,the evaluation of potential new drugs, known as lead compounds, bypharmaceutical companies now requires elaborate, expensive tissueculture and animal trials. An inexpensive sensor and related hardware toallow in vitro and in vivo monitoring of drug kinetics and effectivenesswill have great value to drug companies searching for ways to streamlinesuch lead compound development.

[0068] 1. Entities Sheltering Biodetectors

[0069] The biological components of the biodetector may be contained inor otherwise may be attached to living or nonliving entities thatstabilize the essential interactions. Configuration of these componentsas such results in a micro sensing system capable of detecting smallquantities of ligands with great specificity and sensitivity.

[0070] Living Entitles. Most typically, the biodetector entity is aliving cell which is genetically engineered to comprise all requiredcomponents. Living entities include, but are not limited to,prokaryotes; eukaryotes; viruses; phage; transformed eukaryotic cells,such as transformed lymphocytes and macrophages; and established celllines. Most typically, the entity is a genetically engineered bacterialcell, such as E. coli. Genetically-modified bacteria can be grownrapidly at low cost, thus the advantage of the use of living cells asbiodetector entity is that pools of these biodetectors can be replicatedand grown once the original biodetector is constructed.

[0071] The use of “living” biodetector entities has several advantages.First, it allows the growth of biodetectors at low cost, once thesensors are engineered. Second, a living biodetector can amplify thedetected signal. For example, the binding of one antigen to the surfaceof the bacteria can trigger a series of light-generating substances tobe made, each of which can produce fight in a repetitive manner. Thus,the binding of one antigen that properly stimulates the system canresult in the production of large amounts of photons from one livingbiodetector. Third, the small size of cell-based biodetectors allows forthe binding of a limited quantity of a ligand or target to a pluralityof such biodetectors, further amplifying the total detectable signal andincreasing sensitivity.

[0072] Non-Living Entities. However, abiotic biodetectors may begenerated as well. The biodetector system may be placed in an inanimategel, in abiotic capsules and liposomes and as such be injected into thebody, or mounted on plates. Further, any other entity capable ofpreserving vectoral metabolism such as a lipid bilayer may be employed.

[0073] 2. The Signal-Converting Element

[0074] The signal converting element is composed of an “extracellular”portion selectively binding a specific substance and an “intracellular”portion capable of activating the transducer. Typically, the signalconverting element will be a transmembrane fusion protein composed of anextracellular ligand-binding portion, e.g. an antibody, and anintracellular enzymatic portion, which is activated upon binding of theextracellular portion to a selected target. Accordingly, the signalconverting element is designed to convert the action of recognizing andbinding of a specific substance, i.e., ligand, into an intracellularsignal, resulting in the activation of the transducer component, which,in turn, activates a promoter that drives the expression of the reporterprotein.

[0075] The Ligand-Binding Domain. Substances which may be identified bythe present invention include, but are not limited to, proteins,peptides, sugars, fatty acids, ions, microorganisms, including bacteria,viruses, parasites and fungi. Accordingly, the ligand-binding domain maybe an antibody, an antibody fragment, cellular receptor or any otherligand-binding protein, such as Staphylococcus Proteins A and G, amacrophage Fc receptor, a carbohydrate moiety, or an ion-binding moiety,such as domains from sodium or potassium channels.

[0076] In specific embodiments, the ligand-binding domain is an antibodyor a derivative thereof, including but not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies or fragments (ScFvs), Fab fragments,F(ab′)₂ fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. In particular the monoclonal antibody technology,phage display technology, and the more recent development of techniquesfor expressing functional antibodies in bacterial cells have increasedthe versatility and ease of identifying suitable ligand-binding domainsfor any desired target. The reference of Orum, et al., 1993, NucleicAcids Research 21(19):449 14498 teaches in detail the construction anduse of ScFvs; the reference of Collet et al., 1992, Proc. Nat'l. Acad.Sci. U.S.A. 89:10026-10030 teaches a binary plasmid system forconstructing antibody libraries. For additional details about theexpression of antibodies in-bacterial cells, see, among other places,Huse et al., 1989, Science 246:1275-1281.

[0077] Moreover, the source of the antibody coding regions is notlimited to those cloned from hybridoma cell lines where the specificityof the antibody is known and is monoclonal in nature. Rather, largeantibody libraries may be employed to generate the fusion proteins suchthat a large number of biodetectors for the detection of an indefiniterange of antigens can be generated.

[0078] The Signal Transforming Domain. The signal transforming domainmay consist of an enzyme or active domain of an enzyme that has anynumber of protein modifying functions which may include phosphorylation,dephosphorylation, methylation, acetylation and protease activity. Suchenzymes include protein kinases, phosphorylases, phosphatases, proteinmethylases, acetylases, and proteases, among others. Particularlysuitable signal transforming domains are derived from signaltransduction pathways in bacteria (Parkinson, 1993, Cell 73:857),especially two-component systems. Two component systems consist of atransmembrane “sensor”, typically located in the inner cytoplasmicmembrane, which monitors some environmental parameter, and a cytoplasmicresponse regulator that mediates an adaptive response, usually via analteration in gene expression. Such systems are referred to herein as“sensor/transducer” pairs, where the cytoplasmic regulator is the“transducer”. Activation of the transducer is typically throughphosphorylation by its cognate sensor. E. coli is thought to have about50 such sensor/transducer pairs (Stock, et al., 1990, Nature344:395-400). They include the Omp proteins involved in osmoregulation,proteins associated with nitrogen assimilation and proteins involved inchemotaxis.

[0079] The application of a two-component system to the presentinvention is illustrated with the PhoQ/PhoP system (Miller, et al.,1989, Proc Natl Acad Sci 86:5054-5058; Soncini, et al, 1995, J Bacteriol177:4364-4371). The active domain of the bacterial phosphorylase, PhoQ,is linked in a gene fusion to a region of cDNA encoding antibodydomains. As such, interaction of the expressed fusion protein with thetargeted antigen (ligand) results in a conformational change in theantibody-phosphorylase fusion. This conformational change activates thespecific phosphorylase activity which activates PhoP, a transducerprotein, through a phosphorylation/dephosphorylation event. Active PhoPbinds to and thus activates the PhoN promoter which is used to driveexpression of the reporter operon lux. Of course, one skilled in the artmay practice the invention by similarly adapting and utilizing theproteins from any suitable two-component system.

[0080] Vectors for Expressing the Signal Converting Element. The presentinvention also includes expression vectors useful for making biodetectorsuch as those described herein. A vector according to this aspect of theinvention typically comprises (i) a first DNA fragment encoding anN-terminal leader sequence, (ii) a multiple cloning site (MCS) forinsertion of a DNA fragment encoding an extracellular ligand-specificmoiety, and (iii) a second DNA fragment encoding an intracellular signaltransforming domain. The N-terminal leader sequence can be any bacterialsequence that is effective to direct an expressed polypeptide at leastpart-way through the outer membrane of a bacterial cell. Such sequencesare known in the art, e.g., the N-terminal leader sequence from theexported bacterial protein pectate lyase. The multiple cloning sitecontains at least two adjacent restriction endonuclease sites suitablefor the insertion of a selected sequence, e.g., an EcOR I site and aHind III site.

[0081] In one embodiment, the vector further comprises, between thecloning site and the second DNA fragment, a third DNA fragment encodinga membrane anchor, such as a DNA fragment encoding a modified form of E.coli cell envelope component PAL (Fuchs, et al., U.S. Pat. No.5,591,604, issued Jan. 7, 1997). The vector also typically contains aselectable antibiotic resistance gene (e.g., Amp), and a promoter (e.g.,a T3 or T7 promoter) operably linked upstream of the DNA encoding theN-terminal leader sequence. An exemplary vector according to this aspectof the invention is shown in FIG. 6.

[0082] The vector is useful for the construction of biodetectors, asdetailed herein. DNA fragments encoding antibody fragments can be clonedinto the MCS and expressed as fusion proteins having an N-terminalleader sequence adjacent the antibody fragment(s) adjacent a polypeptidefunctioning as the intracellular signal transforming domain.

[0083] 3. Transducers

[0084] The transducer is activated by the signal converting element uponligand binding. The transducer may be activated by phosphorylation,glycosylation, methylation electron transport, hydrogen transport,carboxylation, dehydrogenation, oxidation/reduction or any otherchemical modification. The transducer may be any molecule that canrecognize and respond to a change in conformation, electrical charge,addition or subtraction of any chemical subgroup, such asphosphorylation, glycosylation, and in turn is capable of triggering adetectable response. In embodiments wherein the the intracellular signaltransforming element is derived from an endogenous signalling pathway,such as a bacterial two-component pathway, the transducer of theinvention is typically the same transducer as is used in the endogenouspathway. Thus, in embodiments which employ an intracellular signaltransforming domain derived from a C-terminal portion of. PhoQ, thetransducer is preferably PhoP that is endogenous to the bacterial cellsheltering the biodetector.

[0085] In specific embodiments of the invention, activation of thetransducer triggers, directly or indirectly, the activation of atranscription control element, e.g., a promoter, to allow expression ofa reporter gene or reporter operon. Transcription and translation of thereporter gene or operon in turn results in a gene product or geneproducts which produces a detectable signal, such as light. However, inalternative embodiments, activation of the transducer may directlyresult in a visible and measurable signal.

[0086] 4. Responsive Elements

[0087] A responsive element according to the present invention istypically a transcription control element or promoter operatively linkedto the reporter gene or operon. In a preferred embodiment, the activatedtransducer binds to the transcription control element or promoter andactivates or turns on the promoter to initiate transcription of thedownstream gene or operon. Regulatable promoters are well known in theart. One embodiment of the invention thus includes promoters and/ortranscriptional activators which control transcription in two-componentsystems. In one specific embodiment, the responsive element comprisesthe phoN promoter, isolated, e.g., as described in Example 2A.

[0088] 5. Reporter Genes and Operons

[0089] A wide range of reporter genes or reporter operons may beemployed, including such which result in bioluminescence, colorimetricreactions or fluorescence. For example, reporter genes may encode forpigments (Bonhoeffer, 1995, Arzneimittelforschung 45:351-356) such asbacterial rhodopsin (Ng et al., 1995, Biochemistry 34:879-890), melanin(Vitkin et al., 1994, Photochemistry and Photobiology 59:455-462),aquorins (Molecular Probes, Seattle), green fluorescent protein (GFP,Clonetech, Palo Alto; Chalfie et al., 1994, Science 263:802-805; Cubittet al., 1995, TIBS 20:448-455), yellow fluorescent protein (Daubner etal., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:8912-8916), flavins,bioflavinoids, hemoglobin (Chance et al., 1995, Analytical Biochemistry227:351-362; Shen et al., 1993, Proc. Natl. Acad. Sci. U.S.A.90:8108-8112), heme (Pieulle et al., 1996, Biochem. Biophys. Acta 1273:51-61), indigo dye (Murdock et al., 1993, Biotechnology 11:381-386),peridinin-chlorophyll-a protein (PCP) (Ogata et al., 1994, FEBS Letters,356:367-371), or pyocyanine (al-Shibib and Kandela, 1993, ActaMicrobiologica Polonica 42:275-280). Alternatively, reporter genes mayencode for enzymes that can cleave a color absorbing substrate such asB-lactamase, luminescent and fluorescent proteins, enzymes withfluorescent substrates, or any other gene that encodes an opticallyactive chemical or that can convert substrate to an optically activecompound. In a further alternative, reporter genes may encodephotoproteins. In each case, the reporter is operatively linked to aninducible promoter which is activated by the active form of thetransducer component.

[0090] In a specific embodiment of the invention, bioluminescentreporters are employed.

[0091] Bioluminescence-Based Reporter Genes And Operons. Several typesof bioluminescent reporter genes are known, including the luciferasefamily (e.g., Wood et al., 1989, Science 244:700-702). Members of theluciferase family have been identified in a variety of prokaryotic andeukaryotic organisms. Luciferase and other enzymes involved in theprokaryotic luminescent (lux) systems, as well as the corresponding luxgenes, have been isolated from marine bacteria in the Vibrio andPhotobacterium genera and from terrestrial bacteria in the Xenorhabdusgenus, also called photorhalodus.

[0092] An exemplary eukaryotic organism containing a luciferase system(luc) is the North American firefly Photinus pyralis. Firefly luciferasehas been extensively studied, and is widely used in ATP assays. cDNAsencoding luciferases from Pyrophorus plagiophthalamus, another species,click beetle, have been cloned and expressed (Wood et al., 1989, Science244:700-702). This beetle is unusual in that different members of thespecies emit bioluminescence of different colors. Four classes ofclones, having 95-99% homology with each other, were isolated. They emitlight at 546 nm (green), 560 nm (yellow-green), 578 nm (yellow) and 593nm (orange).

[0093] Luciferases require a source of energy, such as ATP and NAD(P)H,and a substrate, such as luciferin, decanal (bacterial enzymes) orcoelentrizine and oxygen. The substrate luciferin must be supplied tothe luciferase enzyme in order for it to luminesce. The substrate (e.g.,luciferin or coelentrizine) may be applied externally or supplied byproviding the enzymes for its synthesis. For external application ofluciferin, the substrate may be dissolve n DMSO at a concentration of,e.g., 20 mM-200 mM. A convenient method for providing luciferin is toexpress the biosynthetic enzymes for the synthesis of the substratedecanal. In bacteria expressing these proteins, oxygen is the onlyextrinsic requirement for bioluminescence. In particular, the lux operonobtained from the soil bacterium Xenorhabdus luminescence (Frackman etal., 1990, J. Bact. 172:5767-5773) may be used as reporter operon, as itconfers on transformed E. coli the ability to emit photons through theexpression of the two subunits of the heterodimeric luciferase and threeaccessory proteins (Frackman et al., supra).

[0094] Optimal bioluminescence for E. coli expressing the lux genes ofX. luminescence is observed at 37° C. (Szittner and Meighen 1990, J.Biol. Chem. 265:16581-16587; Xi et al., 1991, J. Bact. 173:1399-1405),which contrasts with the low temperature optima of luciferases fromeukaryotic and other prokaryotic luminescent organisms (Campbell, 1988,Chemiluminescence. Principles and Applications in Biology and Medicine(Chichester, England: Ellis Horwood Ltd. and VCH VerlagsgesellschaftmbH)). Thus, the reporter operon may be chosen according to the natureand the requirements of a specific application. For example, theluciferase from X. luminescence, therefore, is well-suited for use as amarker for studies in animals.

[0095] Luciferase vector constructs can be adapted for use intransforming a variety of host cells, including most bacteria, and manyeukaryotic cells. In addition, certain viruses, such as herpes virus andvaccinia virus, can be genetically-engineered to express luciferase. Forexample, Kovacs and Mettenlieter, 1991, J. Gen. Virol. 72:2999-3008,teach the stable expression of the gene encoding firefly luciferase in aherpes virus. Brasier and Ron, 1992, Meth. in Enzymol. 216:386-396,teach the use of luciferase gene constructs in mammalian cells.Luciferase expression from mammalian cells in culture has been studiedusing CCD imaging both macroscopically (Israel and Honigman, 1991, Gene104: 139-145) and microscopically (Hooper et al., 1990, J. Biolum. andChemilum. 5: r23-130).

[0096] C. Imaging of Light-Emitting Biodetectors

[0097] Light emitting biodetectors may be imaged in a number of ways.Guidelines for such imaging, as well as specific examples, are describedbelow.

[0098] 1. Photodetector Devices

[0099] In one embodiment of the present invention where the signalgenerated by the biodetector is light, an important aspect will be theselection of a photodetector device with a sensitivity high enough toenable the imaging of faint light. Furthermore, in cases where thebiodetector is used in a living subject, the imaging has to be in areasonable amount of time, preferably less than about thirty (30)minutes.

[0100] In cases where it is possible to use light-generating moietieswhich are extremely bright, and/or to detect light-emitting conjugateslocalized near the surface of the subject or animal being imaged, a pairof “night-vision” goggles or a standard high-sensitivity video camera,such as a Silicon Intensified Tube (SIT) camera (e.g., HamamatsuPhotonic Systems, Bridgewater, N.J.), may be used. More typically,however, a more sensitive method of light detection is required.

[0101] At extremely low light levels, such as those encountered in thepractice of the present invention, the photon flux per unit area becomesso low that the scene being imaged no longer appears continuous.Instead, it is represented by individual photons which are bothtemporally and spatially distinct from one another. Viewed on a monitor,such an image appears as scintillating points of light, eachrepresenting a single detected photon.

[0102] By accumulating these detected photons in a digital imageprocessor over time, an image can be acquired-and constructed.Alternatively, the scintillating points can be enumerated and reportednumerically, thus obviating the image reconstruction step and expeditingthe analysis. In contrast to conventional cameras where the signal ateach image point is assigned an intensity value, in photon countingimaging the amplitude of the signal carries no significance. Theobjective is to simply detect the presence of a signal (photon) and tocount the occurrence of the signal with respect to its position overtime.

[0103] At least two types of photodetector devices, described below, candetect individual photons and generate a signal which can be analyzed byan image processor.

[0104] Reduced-Noise Photodetection Devices. The first class constitutesdevices which achieve sensitivity by reducing the background noise inthe photon detector, as opposed to amplifying the photon signal. Noiseis reduced primarily by cooling the detector array. The devices includecharge coupled device (CCD) cameras referred to as “backthinned,” cooledCCD cameras. In the more sensitive instruments, the cooling is achievedusing, for example, liquid nitrogen, which brings the temperature of theCCD array to approximately −120° C. The “backthinned” refers to anultra-thin backplate that reduces the path length that a photon followsto be detected, thereby increasing the quantum efficiency. Aparticularly sensitive backthinned cryogenic CCD camera is the “TECH512,” a series 200 camera available from Photometrics, Ltd. (Tucson,Ariz.).

[0105] Photon Amplification Devices. A second class of sensitivephotodetectors includes devices which amplify photons before they hitthe detection screen. This class includes CCD cameras with intensifiers,such as microchannel intensifiers. A microchannel intensifier typicallycontains a metal array of channels perpendicular to and co-extensivewith the detection screen of the camera. The microchannel array isplaced between the sample, subject, or animal to be imaged, and thecamera. Most of the photons entering the channels of the array contact aside of a channel before exiting. A voltage applied across the arrayresults in the release of many electrons from each photon collision. Theelectrons from such a collision exit their channel of origin in a“shotgun” pattern, and are detected by the camera.

[0106] Even greater sensitivity can be achieved by placing intensifyingmicrochannel arrays in series, so that electrons generated in the firststage in turn result in an amplified signal of electrons at the secondstage. Increases in sensitivity, however, are achieved at the expense ofspatial resolution, which decreases with each additional stage ofamplification.

[0107] An exemplary microchannel intensifier-based single-photondetection device suitable for the practice of the invention is the C2400series, available from Hamamatsu.

[0108] Image Processors. Signals generated by photodetector deviceswhich count photons need to be processed by an image processor in orderto construct an image which can be, for example, displayed on a monitoror printed on a video printer. Such image processors are typically soldas part of systems which include the sensitive photon-counting camerasdescribed above, and accordingly, are available from the same sources(e.g., Photometrics, Ltd., and Hamamatsu). Image processors from othervendors can also be used, but more effort is generally required toachieve a functional system.

[0109] The image processors are usually connected to a personalcomputer, such as an IBM-compatible PC or an Apple Macintosh (AppleComputer, Cupertino, Calif.), which may or may not be included as partof a purchased imaging system. Once the images are in the form ofdigital files, they can be manipulated by a variety of image processingprograms (such as “ADOBE PHOTOSHOP,” Adobe Systems, Mountain View,Calif.) and printed.

[0110] 2. Constructing an Image of Photon Emission

[0111] In cases where, due to an exceptionally bright light-generatingmoiety and/or localization of light-emitting conjugates near the surfaceof the subject, a pair of “night-vision” goggles or a high sensitivityvideo camera was used to obtain an image, the image is simply viewed ordisplayed on a video monitor. If desired, the signal from a video cameracan be diverted through an image processor, which can store individualvideo frames in memory for analysis or printing, and/or can digitize theimages for analysis and printing on a computer.

[0112] Alternatively, if a photon counting approach is used, themeasurement of photon emission generates an array of numbers,representing the number of photons detected at each pixel location, inthe image processor. These numbers are used to generate an image,typically by normalizing the photon counts (either to a fixed,pre-selected value, or to the maximum number detected in any pixel) andconverting the normalized number to a brightness (grayscale) or to acolor (pseudocolor) that is displayed on a monitor. In a pseudocolorrepresentation, typical color assignments are as follows. Pixels withzero photon counts are assigned black, low counts blue, and increasingcounts colors of increasing wavelength, on up to red for the highestphoton count values. The location of colors on the monitor representsthe distribution of photon emission, and, accordingly, the location oflight-emitting conjugates.

[0113] In order to provide a frame of reference for the conjugates, agrayscale image of the (still immobilized) subject from which photonemission was measured is typically constructed. Such an image may beconstructed, for example, by opening a door to the imaging chamber, orbox, in dim room light, and measuring reflected photons (typically for afraction of the time it takes to measure photon emission). The grayscaleimage may be constructed either before measuring photon emission, orafter.

[0114] The image of photon emission is typically superimposed on thegrayscale image to produce a composite image of photon emission inrelation to the subject.

[0115] If it desired to follow the localization and/or the signal from alight-emitting conjugate over time, for example, to record the effectsof a treatment on the distribution and/or localization of a selectedbiocompatible moiety, the measurement of photon emission, or imaging canbe repeated at selected time intervals to construct a series of images.The intervals can be as short as picoseconds (in fast gated cameras) orseconds, to days or weeks with integrating cameras.

[0116] D. Applications

[0117] Specific applications of the biodetectors include the diagnosisof diseases, detection of clinically relevant substances, detection ofenvironmental contaminants, detection of food contaminants. Further, thebiodetectors of the invention will find numerous applications in basicresearch and development.

[0118] Diagnosis Of Infectious Disease. The biodetectors may be used forthe detection of antigens in body fluids, including blood or urine, ortissues and other fluids. Suitable target antigens include, but are notlimited to, bacterial pathogens, viral pathogens, fungal pathogens,serum proteins, lymphokines, cytokines, cytotoxins, interferons, β-2microglobulin, immunoglobulins, peptides, and polypeptides.

[0119] Specific diagnostic tests targeting bacterial pathogens mayinclude, but are not limited to, diagnosis of lyme disease,Streptococcus, Salmonella, Tuberculosis, Staphylococcus, Pseudomonas,Helicobactor, Listeria, Shigella, Proteus, Enterococci, Clostridium,Bordatella, Bartonella, Rickettsia, Chlamydia, Spirochetes, as well asproducts (e.g., toxins) of these and other pathogens. Diagnostic teststargeting viral pathogens may include, but are not limited to, thedetection of retroviruses, such as HIV-1, HIV-1, hepatitis viruses (HBV,HCV, HAV), herpes viruses, including EBV, CMV, herpes simplex 1, herpessimplex II, and HHV-6, alphaviruses, including Japanese encephalitisvirus, Eastern and Western Encephalitis Virus, rotaviruses,Rhinoviruses, influenza viruses, Pox viruses, all known and yet to beidentified human and animal viral pathogens, and unconventional agentssuch as those associated with Alzheimer's and Crutzfeld-Jacob disease(prions). Targeting fungal pathogens may include, but are not limitedto, cryptococcus, histoplasmosis, coccidiodes, and candida.

[0120] Detection Of Other Clinically Relevant Substances. Applicationsof the biodetectors may include the detection of clinically relevantsubstances, such as sugar molecules, fatty acids, or proteins, in bodyfluids, e.g., blood or urine, or tissue. Targeted antigens may includeenzymes indicating the proper function of organs, including lactatedehydrogenase, urea, glucose, and other small molecules, and cytokines.Alpha fetal protein may be targeted for the diagnosis of spinobifida.Certain bacterial species or other microorganisms may be targeted tomeasure their representation in mixed populations such as gut andvaginal flora. An important diagnostic target will be lymphokines forthe diagnosis and prognosis of a range of diseases. With currentmethods, the profile of lymphokines cannot easily be determined,however, it can be expected that its determination will elucidate a widearray of unknown aspects about the relationship of diseases and diseasestates. Further, may be applied to the early, perinatal diagnosis ofgenetic diseases, including cystic fibrosis, sickle cell anemia, Downsyndrome, phenylketonuria, ADA deficiency, thallassemias, growth hormonedeficiency, predisposition of cancer. In addition, the biodetectors mayfind application in the real time monitoring of, e.g., glucose levelsand-drug levels. Finally, biodetectors of the invention may be used intesting for the presence of drugs and/or their metabolites in variousdrug testing applications.

[0121] Agricultural And Veterinary Applications. All above describedmedical applications may be applied to veterinary medicine.

[0122] Detection Of Environmental Contaminants. For example, thebiodetectors may be used for detection of contaminants in water supply.Selected targets may include, but are not limited to Giardia,Cryptococcus, Legionella, Clostridia toxins, Enterobacter, E. coli,protozoans, heavy metals. Further, representation of certain bacteria insoil populations may be measured by the means of the biodetectors; soilmay be screened to track genetically engineered organisms that mighthave been released into the environment.

[0123] Detection Of Food Contaminants. The biodetectors may be employedto identify contaminants in food, including, but not limited to,bacteria, such as Salmonella, Coliforms, Staphylococcus, Clostridium,and fungi.

[0124] Basic Research And Development. The biodetectors will findnumerous applications in basic research and development. Examplesinclude detection system in standard immunoassay, such as Western Blots,ELISA, the determination of lymphokine profiles, the detection of cellculture contamination, including Mycoplasma. Further, the biodetectorswill be useful as detection system in expression assays, for thedetection of cell surface markers, such as CD4, CD8, adherins.

[0125] Abiotic Biodetectors. For certain applications, when antigenicityis an issue (i.e., in vivo) abiotic biodetectors may be desirable.Examples include the in vivo detection and localization of infection,tissue damage and other pathologies. Encapsulation of the biodetectormechanism in generally inert vesicles bilayer or membranes or any otherentity that is non-living and will preserve vectoral metabolism (such asliposomes) in such way that contact with ligands results in light willpermit the use of this system in vivo.

[0126] The following examples illustrate, but in no way are intended tolimit the present invention.

VII. EXAMPLES

[0127] Material and Methods

[0128] Nucleic acid primers (e.g., PCR primers) may be obtained fromOperon Technologies, Alameda, Calif. Primer sequences are designed usingOligo software (Rychlik and Rhoads (1989) Nucleic Acids Res17:8543-8551) and unless otherwise indicated, are derived from publishedsequences available from public sequence databases (e.g., Genbank). DNAsequencing may be carried out on an ABI Prism 310 System (PE AppliedBiosystems, Foster City, Calif.). Unless otherwise indicated, allmolecular cloning techniques and PCR reactions are carried out usingstandard methods (Ausubel, et al., 1998, “Enzymatic manipulations of DNAand RNA”, in Current Protocols in Molecular Biology, pg. 3.0.1-3.19.8;Ausubel, et al., 1998, “The polymerase chain reaction”, in CurrentProtocols in Molecular Biology, pg. 15.0.1-15.8.8). Bacteria aretransformed using standard electroporation or CaCl₂ methods (Ausubel, etal., 1998, “Introduction of plasmid DNA into cells”, in CurrentProtocols in Molecular Biology, pg. 1.8.1-1.8.10).

[0129] The following examples represent approaches which may be employedto link the signal transduction to the expression of a specific gene.

Example 1

[0130] Linking Signal Transduction to the Regulation of a Specific Gene(Approach 1) The following example illustrates one approach which can beused to link the signal transduction to regulation of a specific gene.

[0131] A transposon is constructed to identify promoters that areactivated by ligand binding to surface expressed ligand-bindingmolecules, e.g., antibodies. Promoterless reporter systems have beenemployed for identifying a variety of regulatory sequences in bacteria.Ronald et al., 1990, Gene 90:145-148. The transposon consists of (i) (1)a promoterless operon containing the genes for bioluminescence, (2) aselectable marker (kanamycin resistance gene; Kan), and (3) a negativeregulator (the lambda repressor); (ii) an additional selectable marker(chloramphenicol resistance gene; Chl) expressed by the lambda operator;and (iii) a third selectable marker that is constitutively expressed(ampicillin resistance gene; Amp). Bacterial cells expressing theantibody of interest are transformed with the transposon. Theconformational change in the transmembrane antibody-fusion proteinsignals the activation or chemical modification of the transducer whichis designed to relay that message to the promoter region of the luxconstruct. Positive transformants are selected by determination of theacquired Amp resistance. Cells containing the transposon behindpromoters that are active in the presence of antigen (includingconstitutive expression) will be Kan resistant in the presence ofantigen, and cells containing a transposon behind promoters that are offin the absence of antigen will be Chl resistant in the absence ofantigen. Therefore by passage through a series of growth conditions thedesired transformants that appropriately express luciferase in responseto antigens will be identified. The promoters can then be characterizedand used to construct additional biodetectors.

[0132]FIG. 4 depicts a biodetector generated as described in EXAMPLE 1.As shown in FIG. 4A, in the absence of antigen, the fusion protein doesnot transduce a signal to the promoter which drives expression of thecloned genes encoded by the integrated transposon. Therefore, thephenotype of the proposed E. coli, in the absence of antigen, isampicillin resistant, chloramphenicol resistant, kanamycin sensitive,and not bioluminescent. Ampicillin resistance is constitutivelyexpressed to maintain selection of the integrated transposon.

[0133] When, however, the promoter is turned on by binding of theactivated transducer, which is activated by ligand binding to the fusionprotein, the luciferase operon, the kanamycin resistance gene, and thelambda repressor are expressed. The lambda repressor acts on the lambdaoperator, thereby shutting down the expression of the chloramphenicolresistance gene. In the presence of antigen the phenotype of the cellsis therefore characterized by ampicillin resistance, kanamycinresistance, chloramphenicol sensitivity, and bioluminescence.

[0134] Thus, induction and activation of genes as described abovepermits positive selection for the desired response to antigen. Morespecifically, only those bacterial cells which integrate the describedtransposon at a suitable site in the genome survive the selectionprocedures while nonresponsive bacteria die.

Example 2

[0135] Linking Signal Transduction to the Regulation of a Specific Gene(Approach 2)

[0136] The following example illustrates a second approach which can beused to link the signal transduction to regulation of a specific gene.It employs a fusion protein composed of an antibody heavy chain and asurface protein known to transduce signals for gene regulation, and apromoter that is affected by this signal placed in front of the reporteroperon.

[0137] A. Construction of luciferase reporter construct. The promoterregion of the S. typhimurium phoN gene (Kasahara, et al., 1991, J.Bacter. 173:6760-6765) was amplified by PCR using standard methods(Ausubel, F. M., et al., Current Protocols in Molecular Biology (JohnWiley and Sons, Inc., Media, Pa.) with specific primers (DP1, having thesequence 5′ CTG CTG TCT AGA TTA CTT AGC TAC AGG GAG 3′ (SEQ ID NO: 1),corresponding to bp 5-22 of the sequence having Genbank accession numberX63599; and DP2, having the sequence 5′ GAC CAA GGA TCC CAT AAA GAC TCACTC CGG 3′ (SEQ ID NO:2), corresponding to bp 532-549 of the sequencehaving Genbank accession number X63599). The primers were designed withenzyme restriction sites near their 5′ ends—DPI contains an Xba I site,and DP2 contains a Bam HI site. The PCR products were purified using a“QIAquick” PCR purification column (Qiagen Inc., Valencia, Calif.) andcloned into a “pBlueScript II” vector (Stratagene, La Jolla, Calif.)that contains the luxCABDE operon from P. luminescens. This plasmidconstruct is transformed into E. coli and screened for bioluminescenceunder conditions that activate the PhoP/Q signal transduction system.The phoN-lux construct is then permanently introduced into the E. colibacterial genome by Tn5 transposon insertion using standard techniques(Simon R, et al., 1989 Gene 80:161-169). The construct preferablycontains an antibiotic resistance gene (e.g., ampicillin resistancegene) under the control of a constitutive promoter so that selection ofthe integrated transposon is possible.

[0138] B. Construction of phage-antibody display library. Messenger RNAis isolated from a number of naive mouse splenic lymphocytes andconverted into cDNA using commercially available kits (available from,e.g., Promega Corporation, Madison, Wis.). This pool of cDNA serves asthe template for PCR-amplified antibody genes. A first set of primersspecific for the variable region of the heavy chain and a second set ofprimers for the variable region of the light chain have been previouslydescribed (Orum, et al. 1993 Nucleic Acids Res 21:4491-4498). These areused in separate PCR reactions to generate a large pool of variableregion fragments. By a series of PCR reactions the heavy and light chainfragments are connected to a linker that acts as a hinge region andallows the heavy and light chains to form a single chain variablefragment (ScFv) (Orum, et al., 1993, Nucleic Acids Research21(19):4491-4498. Using a commercially available kit for Ab-phagedisplay (Stratagene, La Jolla, Calif.), these DNA fragments are clonedinto a phagemid vector and transformed into a bacterial host. Phageparticles are rescued with a helper virus and ScFvs are displayed on thesurfaces of the phage in the library.

[0139] C. Antibody selection and screening. Selection of antigenspecific antibodies is accomplished by bio-panning methods (Hoogenboom,H. R, 1997 Trends Biotechnol 15:62-70). The agent (e.g., bacteria,virus, toxin, or purified protein) is attached to a solid support, suchas immunotubes, polystyrene plates, or sepharose columns. The phagelibrary expressing ScFvs is panned over the immobilized antigen, washed,and phage that specifically bind are eluted and amplified. Successiverounds of this selection process increase specificity and affinity.

[0140] D. Construction of PhoQ fusion protein expression cassettes. Avector effective to express and target fusion proteins of PhoQ andselected antibody-fragments PhoQ fusion protein to the cell surface isconstructed. The expression cassette in the vector includes, from the 3′end, the following elements: (i) DNA encoding an N-terminal leadersequence, (ii) a cloning site for the insertion of an ScFv DNA, (iii)DNA encoding a membrane anchor (e.g., PAL), and (iv) a fragmentcomprising the 3′ end of the phoQ gene. The 3′ end of the phoQ gene (thephoQ gene minus the 5′ sensing domain (Miller, 1991, Mol. Microbiol.5:2073-2078)) encodes the active or signal transforming portion of PhoQ,which is capable of activating the transducer PhoP.

[0141] DNA encoding the N-terminal leader sequence from the exportedbacterial protein pectate lyase, is ligated upstream of a cloning sitesuitable for accepting a selected ScFv fragment generated as describedabove. DNA encoding a modified form of E. coli cell envelope componentPAL (Fuchs, et al., U.S. Pat. No. 5,591,604, issued Jan. 7, 1997) isligated downstream of the ScFv fragment cloning site. The 3′ end of thephoQ gene encoding the signal transforming portion of PhoQ is amplifiedby PCR using specific primers and inserted downstream of the PALsequence. The expression cassette is purified and cloned into a vector,containing a selectable antibiotic resistance gene (e.g., Amp), thatallows surface expression of the protein (e.g., the pAP1 vectordescribed in Fuchs, et al., 1997, supra).

[0142] The antibody variable region genes are amplified by PCR from theDNA isolated from specific phage clones. The amplified PCR fragment arepurified and cloned into the cloning site of the PhoQ fusion plasmidconstructed as described above. The plasmid is transformed into thephoN-lux E. coli and antibiotic resistant colonies are screened forlight production in the presence of an antigen specific for theexpressed ScFv fragment.

Example 3 Multiple Biodetectors

[0143] Construction of Cell-Based Sensor Library.

[0144] The molecular techniques detailed above may be applied to thegeneration of a library of cell-based sensors. The library is generatedessentially as described in Example 2D. A mixture of antibody variableregion genes amplified by PCR from either the original lymphocyte cDNAsor from the phagemids are purified and cloned into the cloning site ofthe PhoQ fusion plasmid constructed as described above. The plasmid istransformed into the phoN-lux E. coli and grown on antibiotic-containingmedia. The resulting transformants comprise a library of E. coli whichcan be screened for response to a selected antigen. Cells which light upfor a selected antigen are then selected for use as biodetectors forthat particular antigen.

[0145] Construction of ordered array format. Individual biodetectors, orbiodetectors selected from a library as described above may be arrangedinto arrays to provide a more convenient screening format. Such orderedarrays are capable of screening for a large number of agents with onesample. Ordered arrays may be arranged on a silicon chip or attached viaalginate or similar compounds to a transparent support such as Mylarfilm. Semi-permeable membranes which overlay the arrays may be used toform “envelopes” into which samples (e.g., blood or urine) may beinjected. After a suitable incubation time the envelopes are read in asuitable luminescence reader.

Example 4 Detection of Substances in Solution

[0146] The following is an illustrative assay to detect ligandsincluding viral and bacterial antigens in solutions such as whole bloodand plasma.

[0147] Samples containing the ligand to be detected and quantified arediluted (2 fold serial dilutions) in 96 well plates along with referencestandards. The specific biodetector is added to each of the wells as aviable active cell, and analyzed immediately. Bioluminescent signalsfrom the plate are detected using a charge coupled device (CCD camera)or a luminometer in a 96 well format. Relative bioluminescence from theunknown samples are plotted on a standard curve for quantitation.

Example 5 Detection of Substances on Solid Support

[0148] The following is an illustrative assay to detect substances onsolid supports such as nitrocellulose or nylon membranes, e.g., inWestern blot analyses using specific biodetectors.

[0149] Following transfer of the proteins to a solid support (PVDFImmobilon membrane, Millipore) using standardized procedures, themembrane is dried and transferred to a dish containing the specificbiodetector, as a biologically active cell, in minimal medium or otherclear buffer containing nutrients for bacterial metabolism. After 30minutes incubation at room temperature, the membrane is removed andsealed while still wet in a zip lock or heat sealable plastic bag.Bioluminescent signal from the biodetectors bound to the membrane isdetected using a CCD detector, or other light sensitive detectionmethods. Signals can be quantified using standard image analysissoftware.

Example 6

[0150] Effect of Human Blood on the Light Emission from BioluminescentSalmonella

[0151] As demonstrated in the following example, fewer than ten (10)bacterial cells can be detected with an intensified CCD detector.

[0152] Two fold serial dilutions of Salmonella, strain LB5000, that hadbeen transformed with a plasmid that conferred constitutive expressionof the luciferase operon were plated in duplicate into 96 well plates.Dilutions were made starting with 30 μl of growth medium alone (leftpanel; indicated as LB5000) and with 30 μl of blood (right panel;indicated as LB5000 and 30 μblood) to determine the effects of blood asa scattering and absorbing medium on the limits of detection. Eachdilution and the numbers of colony forming units (CFU) implied fromplating samples from concentrated wells are indicated in FIG. 5. Therelative bioluminescence for each well as determined by analysis of theimage generated by the CCD detector is shown (FIG. 5). The signal in themore concentrated wells was off scale and the numbers are therefore notlinear at higher concentrations.

[0153] All references are incorporated in their entirety.

It is claimed:
 1. A biodetector for the detection of a selectedsubstance comprising: (a) a signal converting element, comprising anextracellular ligand-specific moiety and an intracellular signaltransforming domain, wherein said extracellular ligand-specific moietyselectively recognizes said selected substance, which recognitionactivates said intracellular signal transforming domain; (b) atransducer, wherein said transducer has an inactive and an active formwhich are distinct from each other, and wherein said activatedintracellular signal transforming domain converts said inactive form ofsaid transducer into said active form of said transducer; (c) aresponsive element, wherein said responsive element is activated by saidactive form of said transducer; and (d) a reporter gene operativelylinked to said responsive element, wherein the activated responsiveelement causes expression of the reporter gene to generate a reportergene product, resulting in a detectable signal
 2. The biodetector ofclaim 1, wherein said signal is detected optically.
 3. The biodetectorof claim 2, wherein said reporter gene product is detectable by meansselected from the group consisting of bioluminescence detection,calorimetric reactions and fluorescence detection.
 4. The biodetector ofclaim 3, wherein said reporter gene product is bioluminescence.
 5. Thebiodetector of claim 3, wherein said reporter gene is luciferase.
 6. Thebiodetector of claim 1, wherein said signal converting element is afusion protein where the extracellular ligand-specific moiety and theintracellular signal transforming domain are heterologous to oneanother.
 7. The biodetector of claim 1, wherein said intracellularsignal transforming element is derived from a membrane signaltransmitter.
 8. The biodetector of claim 7, wherein said membrane signaltransmitter is from a bacterial two component regulatory system.
 9. Thebiodetector of claim 8, wherein said membrane signal transmitter isPhoQ.
 10. The biodetector of claim 9, wherein said responsive elementcomprises the phoN promoter.
 11. The biodetector of claim 9, whereinsaid extracellular ligand-specific moiety is an antibody or fragmentthereof.
 12. The biodetector of claim 11, wherein said extracellularligand-specific moiety is a single chain variable fragment (ScFv). 13.The biodetector of claim 1, wherein said biodetector comprises an intactbacterial cell.
 14. The biodetector of claim 13, wherein saidbiodetector comprises a Gram-positive bacterial cell.
 15. Thebiodetector of claim 14, wherein said bacterial cell is selected fromthe group consisting of Streptococcus, Staphylococcus, Listeria,Clostridium, Bacillus, and Corynebacteria.
 16. The biodetector of claim13, wherein said biodetector comprises a Gram-negative bacterial cell.17. The biodetector of claim 16, wherein said bacterial cell is selectedfrom the group consisting of Escherichia, Salmonella, Pseudomonas,Helicobacter, Shigella, Proteus, Bordetella, Neisseria, Haemophilus,Bacteriodes, Vibrio, Brucella, Campylobacter, Klebsiella, and Yersinia.18. A library of biodetectors, comprising at least about 1000biodetectors of claim 13, wherein the extracellular ligand-specificmoiety of each of said biodetectors comprises a different antibodyfragment.
 19. An expression vector useful for making a fusion proteinfor use in a biodetector, comprising (i) a cloning site for insertion ofa DNA fragment encoding an extracellular ligand-specific moiety, and(ii) a first DNA fragment encoding an intracellular signal transformingdomain, wherein said vector is capable of expressing a fusion proteincomprising (a) a polypeptide encoded by a DNA sequence inserted at saidcloning site, and (b) said intracellular signal transforming domain 20.The vector of claim 19, wherein the vector further comprises, betweensaid cloning site and said first DNA fragment, a second DNA fragmentencoding a membrane anchor.
 21. The vector of claim 19, wherein thevector further comprises, upstream of said cloning site, a third DNAfragment encoding an N-terminal leader sequence.
 22. The vector of claim19, wherein the vector further comprises, inserted at the cloning site,a fourth DNA fragment encoding an extracellular ligand-specific moiety.23. The vector of claim 22, wherein the extracellular ligand-specificmoiety comprises an antibody fragment.
 24. The vector of claim 19,wherein the first DNA fragment encodes a polypeptide comprising thecytoplasmic tail of PhoQ.